Method for producing continuous cell lines

ABSTRACT

The present invention relates to a method for production of continuous cell lines comprising providing living cells of an animal or a human, irradiating said cells with UV light, proliferating said cells and selecting multiplying cells as cells of a continuous cell line.

This application is a divisional of U.S. patent application Ser. No.12/390,187, filed Feb. 20, 2009, now U.S. Pat. No. 8,865,450, whichclaims priority to U.S. provisional Patent Application Ser. No.61/067,174, filed Feb. 25, 2008. Both priority applications areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods for producing cell lines.

BACKGROUND OF THE INVENTION

Cell lines have become a valuable tool for vaccine manufacturing. Theproduction of some important vaccines and viral vectors is still done inembryonated chicken eggs or primary chicken embryo fibroblasts. Primaryavian tissue for virus replication is provided by SPF (specific pathogenfree) production plants. SPF derived tissues are expensive and thequality of the supply material is often hard to control. Therefore,inconsistency and shortage in supply are the most predominantdisadvantages of the technologies based on SPF eggs. The same is truefor approaches where primary fibroblast monolayer cultures are used. Tomultiply cell lines indefinitely, the cells need to be immortalized.Most immortalized cell lines currently in use are descendants of cancercells or of fused hybridoma cells. However, the later technology islimited to fusion with myeloma cells. No general technology exists thatcan generate immortalized cells of different types.

SUMMARY OF THE INVENTION

It is an object of the present invention to produce a continuous cellfrom non-continuous cell material. In particular, the goal was toprovide continuous cell lines that have the potential to proliferatewithout the introduction of foreign viral genes.

Therefore, the present invention provides a method for production ofcontinuous cell lines comprising providing living cells of an animal ora human, irradiating said cells with UV light, proliferating said cellsand selecting cells capable to proliferate after at least 20 passages ascells of a continuous cell line.

Such a continuous cell line is culture of cell that can be propagatedand used for the recombinant expression of biomolecules such asproteins, or for the manufacture of viral products such as viralantigens or a whole virus population, in particular for vaccinationpurposes.

Therefore, the present invention also provides a method of producing avirus comprising providing cells of a continuous cell line obtainable bythe inventive method, infecting said cells with said virus, propagatingsaid virus in said cells and collecting said virus.

In another aspect the invention provides a method of producing arecombinant gene product comprising providing cells of a continuous cellline obtainable by the inventive method, transfecting the cells with anucleic acid encoding said gene product, expressing said gene productand, optionally, collecting said gene product.

In a further aspect the invention provides a continuous cell lineobtainable by the method of providing living cells of an animal or ahuman, irradiating said cells with an effective dose of UV light,proliferating said cells and selecting cells capable of proliferatingafter at least 20 passages as cells of said continuous cell line.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the scheme of the UV treatment procedure.

FIG. 2 shows continuous quail cell cultures

FIG. 3 shows the phylogenetic tree, and treatment route of producing acontinuous quail cell line.

FIG. 4 shows a correlation of the UV dosage to the irradiation time withthe set-up used to produce continuous cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides the production of a continuous cell linethrough UV treatment of cells.

A cell line is a population of cells formed by one or more subculturesof a primary cell culture. Each round of subculturing is referred to asa passage. When cells are subcultured, they are referred to as havingbeen passaged. A specific population of cells, or a cell line, can becharacterized by the number of times it has been passaged. The primaryculture is the first culture following the isolation of cells fromtissue. Following the first subculture, the cells are described as asecondary culture (one passage). After the second subculture, the cellsbecome a tertiary culture (passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore, the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but not limited tothe seeding density, substrate, medium, growth conditions, and timebetween passaging. Culturing can be performed by inoculation of a cellmedium, letting the cells grow until a confluent cell culture or acontinuous film is formed by the cells and inoculating a new cell mediumwith a portion of the confluent cells. Nevertheless, passaging is a toolto evaluate the capability to propagate. Normally, cells, includingnon-irradiated cells, isolated from a tissue can be passaged about 10-20times until they reach a state where no further propagation or celldoubling occurs. The cells then enter a senescent state from which nofurther subcultures can be obtained. Contrary thereto continuous celllines are capable to propagate after more than 20 passages, such asafter more then 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 55, 60, 65, 70, 75 or 80 passages. It has now been found by theinventors that such a continuous cell line which can be passagedmultiple times past the 20^(th) passage, in particular immortalizedcells, can be obtained through alteration of cells by UV treatment, i.e.by irradiating these cells with an effective dose of UV light. The terms“effective dose of UV light” according to the present invention shall bethe amount of irradiation needed for transforming the non-continuouscell lines into continuous cell lines. The effective dose of UV lightranges from the minimum dosage required for such transformations to themaximum dosage which is tolerated by these cells without lethalconsequences for the cell culture as a whole. It is clear that above orunder the effective dose limits continuous cell lines cannot beobtained. The skilled man in the art can easily determine optimumeffective dosages for each cell line on the basis of the information andguidance contained herein with routine optimization. The cells may beprimary cells or cells capable of propagation after a few passages.Culturing of the cell lines can be performed with standard cell culturetechniques, such as in T-flask systems or roller bottle systems, or instirred tank or other bioreactor formats. In several embodiments of theinvention, the culture is adapted to and held under serum-freeconditions.

In the present application the term “UV light” means ultravioletradiation having a wavelength of from 10 to 400 nm, in particular 100 to400 nm. The UV light may be selected from the group consisting of UV C(100 to 280 nm), UV B (280 to 320 nm), and UV A (320 to 400 nm). In someembodiments of the invention, the wavelength is between 200 and 300 nm.Photosensitizing agents such as those which intercalate into the DNA andwhich are activated by UV light may be used to enhance the alteringeffect of the UV radiation, although they are not necessary in allembodiments of the invention. In one embodiment of the present inventionthe UV light is UV C having a wavelength of from about 100 to about 280nm. In another embodiment of the present invention the UV light has awavelength of from about 240 to about 290 nm. In another embodiment ofthe present invention about 85% or more of the UV light has a wavelengthof about 254 nm.

Without being bound by any theory it is believed that the UV lightalters the genetic material of a cell, which introduces mutations. Whilesuch alterations can generally be repaired by the cell's repairmechanisms, some alterations might remain. These alterations canintroduce lethal mutations and also alterations which result in cellimmortalization. From UV irradiation experiments an optimal dosage canbe selected which results in a significant portion of cells which areimmortalized and can be cultured. After passaging, it is believed thatonly viable cells which are capable of multiplying are selected, whichare expected to have only minor alterations with at least one alterationwhich results in immortalization. A significant portion of theirradiated cells will not be immortalized but gain differentalterations, leading to apoptotic or necrotic cells. However, inprinciple, only one cell with the alteration inducing immortalization issufficient to obtain a continuous cell culture, as this cell willcontinue to propagate and survive through the multiple rounds ofpassaging as described herein.

The UV light emission may be a continuous form of UV light emission,e.g. mercury lamp technology, or pulsed UV light, e.g. monochromaticlaser technology. The desired UV intensity may be generated by combiningtwo or more lamps. At least two irradiation procedures may be combinedwith a pause in between. The subject matter of the invention encompassesany effective dosage of UV light, i.e. any dosage of UV light whichalters a cell to proliferate continuously. The effective dosage maydepend on a variety of factors which are generally known in the field,e.g. the physical parameters of the UV irradiation chambers, such assize and diameter of the lamp and the chamber, distance between the cellcontaining medium and the UV light source, light absorption andreflection properties of the material of the chamber. In particularembodiments of the invention, the cells are irradiated in a monolayer,one cellular layer on a surface. By the same token, the wavelength andintensity of the UV light as well as the contact time the cell isexposed to the UV light are also critical for the effective dosage.Furthermore, the effective dosage is also influenced by the cell itself,the medium containing the virus and their light absorption properties.In various embodiments of the invention, the effective dosage issufficient to alter at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or100% of cells contained in the sample, and in other embodiments theeffective dosage is sufficient to alter the cells to a level where atleast 10% of the cells are either altered to grow continuously. 10% to90% of the cells may be killed by the irradiation. In certainembodiments of the invention, a sample containing the cells is exposedto an effective dosage of at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65 or 70 mJ/cm². In some embodiments the effectivedosage is up to about 500, 450, 400, 350, 300, 250, 200, 180, 150, 130or 105 mJ/cm². In particular embodiments of the invention, the UV dosageis between about 70 and 105 mJ/cm². In some embodiments, these dosagesare employed by UV C light. The term “about” refers to the property ofcommon UV lamps which do not provide a discrete UV light at a singlewavelength (as in lasers) but have a gauss shaped spectrum also emittinglight in nearby wavelengths. In embodiments utilizing some of theselamps, “about” refers to a deviation of the wavelength value of 10%.

Before or after irradiation or passaging, the cell line is can befurther selected for fulfilling quality control criteria such assterility, free of mycoplasma contamination, free of adventitious viruscontamination, and/or passing the F-Pert test for the presence ofreverse transcriptase activity, as well as other quality controlcriteria used in the art for selecting cell lines for medicalbiotechnology uses. In this sense “free of” is to be understood thatcontaminations are reduced to be below the detection limit of currentquality test procedures. Since the present technology can generatecontinuous cell lines without the use of viral vectors or introductionof retroviruses, the inventive cell lines are often free of anyretroviral activity, as can be tested by an assay for reversetranscriptase activity. However, such retroviral activity may bespecifically introduced into the cell lines of the invention bymolecular engineering techniques for the purposes of, for example,production of viruses or proteins in the cell lines.

The cell line can be of any eukaryotic cell, particularly of a higherorganism, such as in fish, avian, reptile, amphibious or mammal cellsand even insect or plant cells. Some embodiments utilize mammal cellssuch as of hamster, mice, rat, dog, horse, cow, primate, or human; otherembodiments utilize avian cells such as of chicken, duck, canary,parrot, quail, ostrich, emu, turkey or goose. In general, any birdspecies could be a source of avian cells for use in the invention. Insome embodiments, it is advantageous to utilize a less frequentlydomesticated specie (such as quail or emu) to avoid potentialcontamination of stock tissues with viruses prevalent in more commonlydomesticated species (such as chickens.)

The irradiated cells can be of any type of tissue. In some embodimentsthe tissue is derived from an embryo. In many embodiments, a mixedculture of more than one type of tissue is used, as can be obtained bydisintegrate tissue or multiple tissues. In further embodiments thecells are of the umbilical cord of an embryo. The irradiated cells canbe or the tissue(s) can be of or include e.g. endothelial cells,epithelial cells, pluripotent or totipotent stem cells, embryonic stemcells, neuronal cells, renal cells, liver cells, muscle cells, coloncells, leukocytes, lung cells, ovary cells, skin cells, spleen cells,stomach cells, thyroid cells, vascular cells, pancreatic cells, and/orprecursor cells thereof and combinations thereof.

In many embodiments the cells are attached to a surface duringirradiation or during culturing. Culturing on a surface is especiallysuitable for endothelial cells, whereby the cells can be furtherselected for fulfilling further quality criteria such as theircapability to form monolayers, which can be hampered if the UV dosageintroduces too much damaging alteration. On such a surface the cells mayform monolayers. In particular the cells are cultured or irradiated on amicrocarrier. Alternatively the cells may be either irradiated orcultured or both in suspension. Cells which are initially irradiated orcultured on a surface may later be adapted to growth in suspensionculture.

In another aspect the present invention provides a method of producing avirus comprising providing cells of a continuous cell line obtainable bythe inventive method, infecting said cells with said virus, propagatingsaid virus in said cells and collecting said virus.

In the present invention, the viruses to be produced are selected fromenveloped or unenveloped DNA or RNA viruses, with single or double (DNA)stranded genomes, sense or antisense, continuous or segmented. Theviruses may be selected from the group consisting of baculoviruses,poxviruses, adenoviruses, papovaviruses, parvoviruses, hepadnaviruses,coronaviruses, flaviviruses, togaviruses, astroviruses, picornaviruses,retroviruses, orthomyxoviruses, filoviruses, paramyxoviruses,rhabdoviruses, arenaviruses, and bunyaviruses. In some embodiments ofthe invention, the viruses are selected from the group of envelopedviruses, including, flaviviruses, togaviruses, retroviruses,coronaviruses, filoviruses, rhabdoviruses, bunyaviruses,orthomyxoviruses, paramyxoviruses, arenaviruses, hepadnaviruses,herpesviruses, and poxviruses. In other embodiments, the viruses areenveloped viruses such as influenza, including influenza A, B or C, WestNile Virus, Vaccinia Virus, Modified Vaccinia Virus, or Ross Riverviruses. In other embodiments of the invention, the viruses are selectedfrom the group of enveloped RNA viruses, including, flaviviruses,togaviruses, retroviruses, coronaviruses, filoviruses, rhabdoviruses,bunyaviruses, orthomyxoviruses, paramyxoviruses, and arenaviruses. Inparticular embodiments the virus is MVA (modified vaccinia virusAnkara), TBE (tick-borne encephalitis) virus, Yellow fever virus, WestNile virus, New Caledonia virus or an influenza virus.

After the collecting step, the virus can be inactivated by any knownmeans for virus inactivation, e.g. as disclosed in the US publicationnumber 2006/0270017 A1, which is incorporated herein by reference. Inparticular, inactivation can be performed by formaldehyde treatmentand/or UV irradiation, alone or in combination.

In general, serum or serum-derived substances, such as, e.g., albumin,transferrin or insulin, may comprise unwanted agents that cancontaminate the cell cultures and the biological products obtainedthereof. Furthermore, human serum derived additives have to be testedfor all known viruses, including hepatitis viruses and HIV which can betransmitted via serum. Therefore, according to some embodiments of theinventive method, the cells of the cell line are adapted for growth inserum free media, e.g. they are selected for their capability to grow inserum free media. The media may be free of serum or serum fractions, oralso in general blood constituents. Media for these embodiments of theinvention are selected from DMEM/HAM's F12, RPMI, MEM, BME, Waymouth'smedium, in particular an oligopeptide- or protein-free medium asdescribed in the US 2007/0212770 which is incorporated herein byreference in its entirety, or a combination thereof. Said oligopeptidefree medium may be free of blood proteins or oligopeptides with a sizeof more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 amino acids butmay comprise glutathione. The protein-free medium is substantially freeof proteins but may contain proteins produced by the cell lines orproteases. In particular the medium may also comprise a polyamide asgrowth promoting agent and/or be a chemically defined medium asdescribed in the US 2007/0212770. The term “chemically defined” meansthat the medium does not comprise any undefined supplements, such as,for example, extracts of animal components, organs, glands, plants, oryeast. Accordingly, each component of a chemically defined medium isaccurately defined. The chemically defined media are substantially freeof proteins, or cell hydrolysates but may contain proteins produced bythe cell line or proteases. Examples of such media are given in “A guideto Serum-Free Cell Culture”, GIBCO cell culture (2003).

These media, including the serum free medium, the oligopeptide freemedium or the chemically defined medium, may also comprise glutathioneand/or proteases, in particular trypsin such as porcine or recombinanttrypsin prior or after virus inoculation (Klenk et al. (1975) Virology,68: 426-439). Such proteases may also be required during culturing ofthe cell lines since cells attached to a surface by exhibit strong tovery light adherence. Strongly attached cells can be detached byproteases and/or chelating agents such as EDTA (Doyle et al. Chapter 4:Core Techniques, in: Cell & Tissue Culture: Laboratory Procedures,ECACC, John Wiley & Sons, Chichester (1996)). Furthermore the medium, inparticular the protein free medium, may comprise plant or yeasthydrolysates prior or after inoculation. Of course the medium is alsoexpected to comprise proteins or metabolic products produced by theinventive cell lines.

The cell lines obtainable by the inventive method are generallynon-tumorigenic and/or non-carcinogenic. In some embodiments the cellsof the cell lines are tested and selected for to pass quality test suchas the F-pert test.

In a further aspect the invention provides a method of producing arecombinant gene product comprising providing cells of a continuous cellline obtainable by the inventive method, transfecting the cells with anucleic acid encoding said gene product, expressing said gene productand optionally collecting said gene product. The nucleic acid may beDNA, RNA or PNA. In addition to the gene, the nucleic acid may comprisepromoters for expression in the cell, and selection markers.

In a further aspect the invention provides a continuous cell lineobtainable by the method of providing living cells of an animal or ahuman, irradiating said cells with an effective dose of UV light,proliferating said cells and selecting cells capable to proliferateafter at least 20 passages as cells of said continuous cell line. Theinventive cell lines also include the progeny of such produced celllines. In particular the cell line is defined as being obtainable by theembodiments of the method described herein. The obtainable continuouscell lines may have characteristic features such as telomere activity ofspecific caryotypes associated with the UV irradiation necessary toproduce the continuous cell line. In particular embodiments of theinvention the cells of the cell line are non-tumorigenic and/ornon-carcinogenic and in particular also pass quality tests such as theF-pert test.

In particular embodiments the cell line is a cell line deposited at theEuropean Collection of Cell Cultures (ECACC) with the Deposit ReferenceNo. 08020602, 08020603 or 08020604 corresponding to filing cell linenumber QOR2-SF (RE07169), QOR1 CJ07-18/1/F6 and COR CJ0780,respectively. Further inventive cell lines have the characteristicfeatures, like ability to propagate, cell cycle pattern, telomeraseactivity, caryotype, chromosome pattern or telomere length as saiddeposited cell lines and of course being a continuous cell line.

The present invention is further illustrated by the following exampleswithout being limited thereto.

EXAMPLES Example 1 Temporally Different Radiation of Vero Cells with UVLight for Producing Mutants

Materials:

-   -   TC-Vero medium    -   N1-buffer    -   Trypsin (1:10 dilution)    -   Trypsin inhibitor    -   6-well plates, Corning Cat. No. 3516    -   25 cm² T-Flask, Nunc Cat. No.: 163371    -   UV lamp, VL 50 C, 240 nm Grid-Tube, 50 W, company Vilber-Lourmet        Procedure:        Set-up is done in 6-well plates with 1×10⁶ cells/well and 5 ml        of medium volume (in double set-up). A total of 7 plates (each        time 2 wells/plate) is set up.

After 24 h there was a good monolayer culture.

The 5 ml of medium were drained off to 1 ml, and the opened plates wereirradiated with UV light (distance of the plates from the UV lamp=9 cm)

-   -   plate A: 15 min    -   plate B: 30 min    -   plate C: 45 min    -   plate D: 60 min    -   plate E: 90 min    -   plate F: 120 min    -   plate G: control, no irradiation        After irradiation, the cells of both wells are trypsinized (1 ml        Trypsin+0.5 ml trypsin inhibitor/well), wherein the cells of the        1^(st) well are used for determination of cell count (CC) and        viability, and the cells of the 2^(nd) well are passaged in 25        cm² roux with 10 ml of medium.

Test Irradiation TCC/well Bürker- Türk no. time [×10⁶] viab. [%] A 15min 1.50 60.8 B 30 min 1.25 27.9 C  45 min. 1.15  5.6 D 60 min 0.95 23.6E 90 min 0.55 not determined F 120 min  0.30 not determined G control1.25 94.2T-flask 25 content was trypsinized, the TCC and viability is determinedusing Cedex:

Test TCC/Roux Viab. no. [×10⁶] [%] Microscopic picture A 0.80 23.2spheroidal cells, no adherence B 0.60 18.8 cells in the supernatant, noadherence C 0.60 34.4 individual cells in the supernatant, no adherenceD 0.50 25.0* only cell debris left E 0.50 22.7* only cell debris left F0.40 11.1* only cell debris left G 1.80 96.6 good monolayer, 95-100%*the actual values are lower since the cell count in the Cedex is toolow for correct cell-count determination!!!

Example 2 UV Irradiation of Avian Cells

The aim of this study was to investigate the potential use of UV-lighttreatment as a tool for the generation of continuous cell lines suitablefor vaccine production.

Primary chicken and quail embryos were used as starting material forproduction of initial primary monolayer cultures. Quality controlledcell cultures derived therefrom were used for derivation procedure basedon the UV light exposure.

Exposure of primary cells to UV light (254 nm). The continuous cell linewas developed from primary cells of bobwhite quail or chicken embryos bymeans of UV irradiation.

The detailed course of development of the cell line derived from theprimary cells of quail embryos up to the production of safety banks isillustrated in FIG. 3 in the form of a phylogenetic tree.

As starting material for UV irradiation, in each case one ampoule of thefirst evaluation cell banks (chicken, Japanese quail and bobwhite quail)which originate from a cell preparation of the chicken embryos, embryosof the Japanese quail and of the bobwhite quail (mixed culture ofdisintegrated complete embryos) was thawed.

The set-up for UV irradiation was done in 6-well plates with a cell seedof 1×10⁶ cells/well and 5 ml of medium volume. TBE medium (FSME) with 5%of FBS and antibiotics (penicillin, streptomycin and gentamycin) wereused as medium. A total of 7 plates with 2 well/plate each was set up.After 24 h, a uniform monolayer culture could be observed in the wells.For irradiation of the cells, the 5 ml of medium were drained off to 1ml and the opened plates were irradiated with UV light in the laminarflow bench as follows. The distance of the plates from the UV lamp was 9cm. A UV lamp of the company Vilber-Lourmet (VL 50 C, 240 nm Grid-Tube,50 W) was used as UV light source.

-   -   plate A: 0.5 min    -   plate B: 1 min    -   plate C: 2 min    -   plate D: 3 min    -   plate E: 4 min    -   plate F: 5 min    -   plate G: control, no irradiation

After irradiation, the cells were trypsinized in the wells (1 ml trypsin1:10 diluted with N1 buffer), wherein 1 ml of the cell suspension (atotal of 6 ml) was used for determination of CC and viability, and theremaining cells were passaged with 5 ml of medium in 25 cm² roux. Theresults are summed up in the table of this example.

During the first culturing period (about 25-35 days) there were onlymedia exchanges, and morphology and adherence of the cells wereoptically assessed in the individual tests. Only after island formationof the adherently growing cells in the T-25 flasks had been observed,the cells of tests A-E were trypsinized and transferred to 6-well plates(smaller surface than T-25 flasks) in order to promote a homogeneous,adherent cell colonization. From this point in time, about K40-K50, thecells that had reached a confluence of 80-100% were further passaged inT-25 and T-75 flasks every 6-9 days and set up in 1-2 safety ampouleswhich served as starting material for producing the evaluation cellbanks (about 10 ampoules). Trypsinization and passaging of said cellpopulations is described in Example 3.

Preparations Used

Medium:—TBE medium (FSME)+5% FBS+mixture of antibiotics(penicillin/streptomycin 100 mg/l and 50 mg/l gentamycin)

-   -   TBE medium (FSME)+10% FBS    -   TC Vero medium+10% FBS    -   N1 buffer    -   Gamma trypsin    -   DMSO company Sigma        Abbreviations: CC . . . cell count, T-25/75/175 . . . 25/75/175        cm² T-flasks,

TABLE Cell counts and viabilities of the individual tests afterirradiation Test Irradiation CC/ml Bürker-Türk TCC/well no. time [×10⁶]viab. [%] [×10⁶] A 30 sec 0.75 87.3 0.15 B 1 min 0.75 79.5 0.15 C 2 min0.80 84.1 0.16 D 3 min 0.70 90.4 0.14 E 4 min 0.80 80.0 0.16 F 5 min0.70 * 0.14 G control 0.75 84.1 0.15 * not determined

Due to the similar cell-count values and viabilities of the individualtest set-ups A-G, no significant difference could be shown with respectto UV-irradiation time of the cells. This is the reason why morphologyand adherence of the cultures compared were assessed nearly daily torecognize particularities.

From all test set-ups A-F, the cell population from set-up E showed thebest properties of a continuous, adherently growing cell line, such ashomogeneous cell structure, culturing in different T-flasks, constantcell growth after several passages, capability for cryoconservation andsuitability for virus propagation (e.g. MVA virus).

In the case of quail cells, the cell population from set-up F could notbe successfully cultured. Reduced cell growth with inhomogeneouscell-lawn formation (large wholes) could be observed after more than 6passages with the cells (test G) which had not been irradiated with UVlight. From passage 16 on, the cells lost their division capability andcould not be cultured any longer. All in all, similar results could bereached with quail and chicken cell tests.

Example 3 Trypsinization and Passaging of Cells

Trysinization and passaging of the adherently growing quail cells weredone in a passaging scheme similar to that usually used for Vero cells.After pouring off the culture medium, a washing step is performed withN1 buffer, thereafter, the culture is covered with layer(s) of thecorresponding amount of gamma trysin, diluted 1:10, and is incubated ata temperature (with 6-well plates and T-25 (T-25 . . . 25 cm² T-flasks)room temperature is sufficient) of 37° C. until the cells detach fromthe culturing vessel (by soft knocking). Addition of the trypsininhibitor to stop the effect of trypsin is not necessary due to the FBScontained in the culture medium. Subsequently, the cells are transferredto a new culture medium and are divided up into further culturingvessels in correspondence with the respective splits, and are, again,left to grow.

The following table indicates the amounts used during trypsinization.

Gamma trypsin N1 (1:10 diluted culturing vessel buffer with N1 buffer)6-well plate  2 ml 1 ml 25 cm² T-flask  5 ml 1 ml 75 cm² T-flask 10 ml 1ml 175 cm² T-flask  20 ml 2 ml

Example 4 UV-C Dosimetry for Cell Immortalization with the UV Lamp VL 50C

The dosage to obtain continuous cell lines with UV irradiation wasmeasured. The dosimetry set up was similar to the set up for celltreatment. The radiation with UV-C light causes a transformation ofpotassium iodide and potassium iodate dissolved in buffer solution intobrown-yellow tri-iodide. Tri-iodide has its absorption maximum at 352 nmand can be measured quantitatively in a spectral photometer. Thisprinciple allows to measure the UV dosage applied during cell monolayerexposure depending on exposure time. Therefore, based on measurements in6-well plates, an exposure time of from 0.5 to 5 minutes corresponds toan UV dosage of from 20 to 120 mJ/cm² (FIG. 4).

Dosimetry is done as precisely as possible, as is the cell-line test. Ineach case 1 ml of the model solutions with absorption coefficients (367mn) of about 2.5/cm, 4.5/cm and 7.5/cm is irradiated in one well of the6-well plate. Each model solution is irradiated 6 times. Irradiationtimes=30 sec, 1 min, 2 min, 3 min, 4 min and 5 min. In order to find outthe exact dosage for the respective irradiation time, the OD (253.7 nm)of the medium used is determined.

Materials Used:

-   -   portable UV lamp, VL50 C, 254 nm, 50 W, company Vilber-Lourmat    -   Spectral photometer, company Therma, Device No.: PA5007-012MM    -   6-well plate    -   Boric acid 99.9%, company Riedel-de Haen, Lot No.: 60460    -   NaOH pellets, company Baxter, Lot No.: 318608    -   PVP K17 PF (polyvinyl-pyrrolidon Collidon K17), company Basf,        Lot No.: 30408609T0    -   Potassium iodide, company Sigma Aldrich, Lot No.: P2963-500G    -   Potassium iodate, company Merck, Lot No.: K32577451622    -   TC VERO medium (VT), Charge: ORSFVTC0700401    -   WFI water, company Baxter, PP2

Three model solutions are prepared in sufficient amounts.

TABLE 1 Composition of the model solutions Reagent Model solution 1Model solution 2 Model solution 3 Boric acid 6.18 g/l in Aqua dest.dissolve NaOH pellets desired value pH: 9.15; about 2 g/l PVP K17 PF2.414 g/l Potassium 1.41 g/l 2.57 g/l 4.30 g/l iodide purest Potassium 0.3 g/l 0.55 g/l 0.92 g/l iodate purest

The model solutions can be stored in a dark place until they are usedbut at least up to 47 days.

60 ml each are taken from model solutions 1, 2 and 3 to produce acalibration curve. Protected from incident light, these samples are sentto IBC which establishes the calibration curve.

100 ml each are transferred from model solutions 1, 2 and 3 into Schottflasks and are protected from incident light. The portable UV lamp VL 50C ist placed on a framework. The distance between the table plate andthe bottom side of the portable UV lamp is 9 cm. The portable UV lamp isadjusted such that the filter points to the table plate (i.e.downwards).

The portable UV lamp is turned on 30 minutes before it is used.

The 3 Schott flasks with the 100 ml of model solutions 1, 2 and 3,pipettes, pipettboy, an empty Schott flask and three 6-well plates areprepared. 1 ml of model solution 1 is pipetted into the left upper wellof a 6-well plate. This well is placed below the portable UV lampwithout a cover such that it is positioned centrally below the filter.After 30 sec of irradiation, the well is quickly removed from itsposition below the portable UV lamp. 370 μl of the irradiated 1-mlsolution are transferred to a thin-layered silica cuvette and the OD367nm is determined within 5 minutes. The same is measured three times andrecorded. The mean value of these 3 values is determined. If a valuemeasured is beyond the calibration region of the photometer,correspondingly, a cuvette with a different layer thickness will beused. The supernatant in the well is sucked off and discarded.

These steps are repeated for all irradiation times. Based on theobtained curve functions and OD (253.7 nm) of the VT medium, therespective UV dosage [mJ/cm²] is calculated far from 30 seconds to 5minutes. The results are presented in the following table.

TABLE UV dosage calculated based on the respective curve functions:irradiation time 30 seconds irradiation time 3 minutes potential curvefunction y = 21.767x^(−0.1945) potential curve function y =147.31x^(−0.5363) A 253.7 VT medium (=x)  4.01 A 253.7 VT medium (=x) 4.01 UV dosage [mJ/cm²] 16.61 UV dosage [mJ/cm²]  69.95 irradiationtime 1 minute irradiation time 4 minutes potential curve function y =56.953x^(−0.4709) potential curve function y = 212.7x^(−0.5159)  A 253.7VT medium (=x)  4.01 A 253.7 VT medium (=x)  4.01 UV dosage [mJ/cm²]29.61 UV dosage [mJ/cm²] 103.90 irradiation time  2 minutes irradiationtime 5 minutes potential curve function y = 98.154x^(−0.4322) potentialcurve function y = 264.53x^(−0.5377) A 253.7 VT medium (=x)  4.01 A253.7 VT medium (=x)  4.01 UV dosage [mJ/cm²] 53.86 UV dosage [mJ/cm²]125.36

As can be seen from this table, the curve function of the dosage isy=24.09x+4.3125. X is the irradiation time in minutes and y is thedosage in mJ/cm² (FIG. 4).

Example 5 Virus Production in Continuous Cells

MVA, r-MVA, TBE and Influenza were propagated in continuous quail cells.Roller bottle cultures of quail cells were established as describedabove. Cultures were infected with (GMP) MVA, TroVax, TBE and Influenzavirus. A MOI was chosen according to the current MVA production process.Viral products were harvested after 3 to 4 days. AS culture mediumTC-Vero 10% FBS was used during incubation at incubation 32° C.

Infect.: carried out with 10 ml after 1 h at final volume (60 ml)

TBE: 50 μl virus

MVA: 250 μl virus

New Caledonia (NC): 50 μl

Abbreviation: KXX . . . day of culture XX

Taking of samples: 3×1 ml sample, NOVA, NaBr— with NC, HA,microphotography

TBE

glucose glutamine lactate NH4 CO2 virus titer TBE-Elisa CPE TBE-HA day[g/l] [g/l] [g/l] [mg/l] pH [%] lg pfu/ml μg/ml % HAU/200 μl 1 X X X X XX X X 0 X 2 2.83 0.31 0.33 21 7.36 5.8 neg 0.02 0 16 3 2.71 0.27 0.4 267.32 6 6.34 0.08 0 16 4 2.47 0.2 0.59 47 7.36 5.1 6.87 0.21 0 64MVA

glucose glutamine lactate NH4 CO2 TCID₅₀ CPE day [g/l] [g/l] [g/l][mg/l] pH [%] titer % 1 X X X X X X X X 0 2 2.64 0.31 0.44 20 7.31 6.1 XX 0 3 2.6 0.27 0.58 23 7.26 6.1 X X 0 4 2.36 0.21 0.74 43 7.35 4.97.02e8 V/ml X 0New Caledonia

glucose glutamine lactate NH4 CO2 NaBr CPE day [g/l] [g/l] [g/l] [mg/l]pH [%] mm HA % 1 X X X X X X X X 20 2 2.76 0.3 0.34 21 7.32 6.2 X 3 =8HAU 70 3 2.82 0.26 0.38 25 7.34 5.7 0 5 = 32HAU 100 4 2.63 0.19 0.48 457.41 5 0.00E+00 5 = 32HAU 100Control

glucose glutamine lactate NH4 CO2 NaBr day [g/l] [g/l] [g/l] [mg/l] pH[%] mm HA CPE % 1 X X X X X X X X 0 2 1.78 0.16 1.24 31 7.03 6.3 X X 0 31.24 0.12 1.42 34 6.87 6.9 X X 0 4 1.52 0.11 1.61 53 6.94 4.9 X X 0

Virus titer achieved for MVA and r-MVA grown in roller bottleexperiments:

Virus TCID₅₀/ml MVA 8 × 10⁸ r-MVA (TroVax) 9 × 10⁸

Virus titer achieved for TBE and Influenza grown in roller bottleexperiments:

Virus Titer (log pfu/ml) HA (HAU/50 μl) CPE (%) TBE 6.9 64 100 NewCaledonia not determinated 32 100

Example 6 F-Pert Assay of Different Cell Cultures

The F-Pert assay allows to detect reverse transcriptase activity by PCRand is necessary for safety validation. Different cultures (Vero (neg.control), primary chicken (pos. control), continuous quail andcontinuous chicken cells) were prepared according to the same procedure.Culture supernatants were harvested and processed for F-Pert qualitycontrol testing

Results F-Pert Testing

Cell Culture F-Pert Vero (control) negative CEC (primary chicken cells)positive quail cells (4 different cultures) negative chicken cells (2different cultures) negative

The invention claimed is:
 1. An immortalized avian cell line depositedat the European Collection of Cell Cultures (ECACC) selected from thegroup consisting of: (a) a cell line with deposit reference number08020602; (b) a cell line with deposit reference number 08020603; and(c) a cell line with deposit reference number
 08020604. 2. The cell lineof claim 1 with deposit reference number
 08020602. 3. The cell line ofclaim 1 with deposit reference number
 08020603. 4. The cell line ofclaim 1 with deposit reference number 08020604.